Electrochemical nano-biosensor systems are popular in the industrial field, along with evaluations of medical, agricultural, environmental and sports analysis, because they can simultaneously perform qualitative and quantitative analyses with high sensitivity. However, real-time detection using an electrochemical nano-biosensor is greatly affected by the surrounding environment with the performance of the electron transport materials. Therefore, many researchers are trying to find good factors for real-time detection. In this work, it was found that a composite composed of graphite oxide/cobalt/chitosan had strong stability and electron transfer capability and was applied to a bioelectrochemical nano-biosensor with high sensitivity and stability. As a mediator-modified electrode, the GO/Co/chitosan composite was electrically deposited onto an Au film electrode by covalent boding, while glucose oxidase as a receptor was immobilized on the end of the GO/Co/chitosan composite. It was confirmed that the electron transfer ability of the GO/Co/chitosan composite was excellent, as shown with power density analysis. In addition, the real-time detection of D-glucose could be successfully performed by the developed nano-biosensor with a high range of detected concentrations from 1.0 to 15.0 mM. Furthermore, the slope value composed of the current, per the concentration of D-glucose as a detection response, was significantly maintained even after 14 days. Nano-biosensors are analytical devices composed of biological materials with physicochemical detectors. Due to the capacity to sense many biological reactions, a nano-biosensor can be applied in various areas, such as medical care, environmental protection, food safety, and even industrial bioprocessing monitoring ,2,3. As Many applications of a nano-biosensor have been reported in the medical and food areas , while t2) forms cobalt hydroxide (Co(OH)2). Cobalt hydroxide has well-defined electrochemical redox activity [A proper electron transfer mediator immobilized on an electrode significantly affects the bioeletrochemical detection of nano-biosensors. Generally, layer- or matrix-structured materials such as graphite-based nanocomposite films are employed for electrode modification as mediators to improve electron transfer and to finally enhance electrochemical sensing ,8,9. In activity . Cobalt activity . The intIn this study, the electron transfer mediator immobilized on a film-type Au electrode was modified by GO/Co/chitosan deposition in the process of manufacturing a bioelectrochemical nano-biosensor for the detection of D-glucose. The redox behavior and electrode transfer properties of the established electrode were determined by cyclic voltammetry (CV) and electrical impedance spectroscopy (EIS). Electrochemical measurements were carried out with a broad range of glucose concentrations to investigate the sensing mechanism ,14. The 4, H2SO4, Cobalt (II) chloride hexahydrate (CoCl2·6H2O), chitosan with medium molecular weight, glucose oxidase , covalent bonding agents as N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich reagent Co. . Ammonium hydroxide (NH4·OH), anhydrous dextrose , and potassium permanganate were purchased from Samchun Pure Chemical Company . Deionized (DI) water with a resistivity of 18.1 MΩ·CM was produced using an ultra-pure water system  purifier system.Graphite powder (synthetic < 20 µm), KMnOGold (Au) film was approximately cut to have an area of 10 × 10 mm. It was then cleaned with alcohol for preparing an Au film electrode. The mediator consisting of GO/Co/chitosan was prepared as a mixed solution, which was then electro-deposited onto the surface of the Au film electrode according to a previous method ,16. AfteGox was immobilized on the mediator-modified electrode. Enzyme immobilization was carried out with 1 g/L of Gox in phosphate buffer  containing 20 mM of EDC and 5 mM of NHS as covalent-bonding reagents. After 4 h of immobilization re-action at 4 °C, the modified electrode was stored at 4 °C for 4 h before utilization . Both siThe surface of the GO/Co/chitosan-modified Au film electrode was observed with a field emission–scanning electron microscope (FE–SEM). Energy dispersive X-ray spectrum (EDS) and elemental mapping images of the synthesized composite nanosheets of the film electrode surface were obtained at a high-vacuum mode using a FEI Quanta FEG 250 field FE-SEM with ThermoFisher Quantax EDS attachment. EDS was recorded with a FEI Quanta FEG 250. The mediator-modified Au film was observed by Fourier transform infrared spectroscopy  to confirm functional groups on the electrode surface after modification.The Gox/GO/Co/chitosan/Au film electrode developed in this study served as the working electrode of a three-electrode system, in which a bronze-based gold electrode and commercial platinum electrode were used as the working electrode and counter electrode, respectively. With various glucose concentrations (from 1 to 15 mM), cyclic voltammetry (CV) measurements were carried out in 0.1 M phosphate buffer from −0.8 to 0.2 V with a scan rate of 0.1 V/s. Impedance spectroscopy (EIS) was measured at a frequency from 0.01 Hz to 10 kHz.To further study the performance of a developed film-type biosensor, glucose sensing of a commercial beverage was demonstrated by CV measurement. The sample of the commercial beverage was diluted 10 times with DI water. This real case CV was detected with the VersaSTAT 3 electrochemical analyzer as described previously . Scheme The surface of the Au film electrode modified with a GO/Co/chitosan composite with element distribution was analyzed by FE-SEM . Accordi−1 with stretching vibration of around 3695 and 3073 cm−1 [−1 with stretching vibration from 3666 to 3023 cm−1, respectively. Meanwhile, the absorption peak of C-O-H stretching presented at 1201 cm−1, as shown in −1 [−1 was ascribed to C-O stretching vibration of the C-O-H, CO-C, and CH2OH rings [−1, the spectrum of the chitosan complex was observed at 1595 cm−1, and the carbonyl C=O was reported to have been stretched at 1667 or 1746 cm−1 [−1 after deposition of the chitosan complex. These changes of FT-IR results have been reported for a hybrid complex with two or more polymer mixtures, for which characteristic spectra peaks are influenced by the reflection of physical blends and chemical interactions [There are several picks of atom components at the surface of the modified electrode in GO/Co/chitosan, as shown in the EDS graphs in 073 cm−1 . The FT-wn in −1 . The broOH rings ,20. In a746 cm−1 . Similarractions . The FT-To establish a biosensor for glucose detection, Gox was immobilized on the mediator-modified Au film electrode. Nyquist plots of the Au bare and modified electrodes after enzyme immobilization were prepared to investigate the electrical properties of the prepared electrodes. EIS results of the modified electrode with immobilized Gox are magnified as a red curve compared with those of the bare electrode a,b. The 2. The reduced FADH2 then releases two hydrogens, while hydrogen peroxide (H2O2) is produced. Finally, the H2O2 donates two protons and two electrons, as shown below:As a key factor of the biosensor, redox reaction with immobilized Gox on the electrode directly influenced electron productivity. Thus, it was necessary to determine the mechanism of Gox catalysis in this electrochemical application. Cyclic voltammograms (CVs) of the developed biosensor were obtained with designated potentials to investigate reversible and irreversible oxidation on the electrode. The CV results showed oxidation peaks from −0.2 to 0.2 V and reduction peaks from −0.8 to −0.6 V. The oxidase peaks of CV results for different D-glucose concentrations could be obviously distinguished, confirming that efficient redox reactions occurred on the Gox-immobilized electrode. D-glucose is oxidated to be gluconolactone, while flavin adenine dinucleotide (FAD) as a cofactor of Gox is reduced to be FADH2) could be widened during its reaction with glucose, in which hydrogen peroxide was selected as a substrate to combine with a reductive enzyme rather than to oxygen [2O2 in the redox reaction system directly resulted in decreased reaction of its oxidation to O2, which significantly reduced electron generation from Gox catalysis. Therefore, with an established time, the current value of the oxidation peak might be decreased due to more Gox (FADH2) moving toward an inactive state which could not bind to glucose. To further investigate the sensing behavior of this biosensor, the CV results of the current peak values with various glucose concentrations were obtained. After 14 days of fabrication, a linear function of current peak was obtained: |Ic|P = 3.17 (μA/mM) × C + 0.044 μA (correlation coefficient: 0.9999). This high coefficient indicated a high accuracy of the biosensor for glucose detection. In addition, the sensitivity of this Au film electrode-based biosensor was determined to be 0.14 μA/mM‧cm2 with a detection limit of 2.7 mM. Both sensitivity and glucose-sensing limitation of this biosensor were significantly improved compared to the reported biosensors using immobilized Gox on the Au electrode [It was confirmed by CV cycle with a hydrogen peroxide pathway that the developed GO/Co/chitosan-based nano-biosensor enables the transfer of two elections using D-glucose. The formation of Co nanorods in the GO/Co/chitosan composite has been reported with significant promotion for electron transfer at the active site of Gox ,13,22,23o oxygen ,25,26. Alectrode . This faThe cell potential and power output detection are shown in According to the official composition table, sport beverages have various compositions, such as multiple sugars, citrus flavor, citric acid, sodium citrate, sodium chloride, potassium chloride malic acid, calcium lactate, and glucono-delta-lactone, which can influence glucose sensing ,31,32. W2, with a detection limit of 2.7 mM. The stability of the modified electrode was confirmed. Glucose concentrations could still be distinguished at 14 days after the biosensor was established. Additionally, practical glucose detection for a commercial beverage was demonstrated with this developed film-type biosensor. The results of this study suggest that the Gox-based Au-film working electrode is a promising candidate for a miniaturized glucose biosensor.An Au film electrode was created with immobilized Gox on an EDC/NHS bonding GO/Co/chitosan-modified surface to establish a glucose biosensor. The response of the Au−Gox electrode in the absence of the Gox enzyme was significantly suppressed compared to the response in the presence of the enzyme, suggesting that a hydrogen peroxide pathway dominated our Au−Gox electrode. Modification of the GO/Co/chitosan on the surface of the Au film electrode was analyzed by SEM and FT-IR. The hydrogen peroxide pathway of electron transfer dominated in immobilized Gox, which was confirmed by a significantly facilitated CV response compared to the bare electrode. Based on the CV responses of the anodic peak current, the linear detection range of glucose concentration was found to be between 1 and 15 mM. The sensitivity of this glucose biosensor was determined to be 0.14 μA/mM‧cm


In this study, we investigated the transport mechanism of immune-active peptide fragments isolated from casein gastrointestinal hydrolysates via a Caco-2 monolayer. The casein gastrointestinal hydrolysates could stimulate B-lymphocyte proliferation and reduce the TNF-α level. Then, we identified the bioactive peptide fragments derived from casein gastrointestinal hydrolysis using LC-MS/MS. Our results demonstrated that the transport mechanism of five immune-active peptides at the cell level was bypass transport. In addition, the majority of peptide RYPLGYL was transported through the monolayer cell membrane as an intact form for playing immune-active functions. The KHPIK and FFSDK were mainly degraded into small fragments, except for a small amount passing through Caco-2 cells in an entire form. Overall, these results suggested that casein or its immune-active peptides might play a role in regulation of the intestinal immune system. Therefore, the physiological activity and function of casein after gastrointestinal hydrolysis need to be explored. Recently, it has been shown that some peptides can be absorbed into the circulatory system in a form without being decomposed into amino acids, providing a molecular biological basis for the research and application of immunocompetent peptides [in vivo and in vitro experiments. The in vivo experimental results are more reliable for complex environments, similar to human beings. However, because of the heavy workload, long experimental periods, and the limitation of individual differences in experimental models, in vivo methods are the mainly approaches for experiments, compared to in vitro [in vitro simulated gastrointestinal digestion were similar to that in in vivo experiments [in vitro conditions, including temperature, pH, hydrolysis time, appropriate protease, or combining different proteases for enzymolysis, can also help to obtain robust results.The protein content in milk is high, accounting for about 3.3–3.5% of the total mass, whereas casein accounts for 80% of the total protein in milk . Casein in vitro . Bohn anin vitro the effect of β-CN(193-209) on the cellular function of macrophages. The results showed that this peptide could enhance the expression of compatibility complex II antigen, the main component in macrophages, stimulating cytokines release and improving the phagocytosis of macrophages [Studies have shown that casein contains a varieties of amino acid sequences with immunocompetent activities in their inactive form . After hrophages . Moreoverophages . Howeverin vivo could be investigated on the Caco-2 cell model [Food functional factors can exert their biological activity only if being absorbed and reaching the target organs. The critical point of this process is to resist the hydrolysis of enzymes in the gastrointestinal tract and be absorbed entirely by small intestinal epithelial cells . Howeverll model . Some stll model .Here, bovine casein was digested by the simulating human gastrointestinal environment to detect immune activity and identify the immune-active peptides through LC-MS/MS. In addition, the absorption forms and mechanisms of immune-active peptides were investigated using the Caco-2 cell model.B-lymphocyte , TNF-αEnzyme-linked immunosorbent assay (ELISA) kit ; Arg-Tyr-Pro-Leu-Gly-Tyr-Leu (RYPLGYL), Lys-His-Pro-Ile-Lys-His (KHPIKH), Lys-His-Pro-Ile- Lys (KHPIK), Phe-Phe-Ser-Asp-Lys (FFSDK), Tyr-Gly-Gly  Human colorectal adenocarcinoma Caco-2 cell line were purchased from the Wuhan species collection center . DMEM high glucose medium, fetal bovine serum, penicillin-streptomycin , non-essential amino acids, and trypsin-0.02% EDTA digestion solution were obtained from Hyclone . Gly-Pro was provided from Shanghai Top Technology Co., Ltd. . phenylarsine, sodium deoxycholate, MK-571 sodium salt, sodium azide, verapamil, fluorescent yellow, dimethyl sulfoxide (DMSO), and 3,4,5-dimethylthiazol- 2,5-diphenyl-tetrazolium bromide (MTT) were purchased from the Sigma Chemical Co. . In total, 12-well Transwell plates and other types of cell culture plates were purchased from Corning Incorporated. All other reagents were of analytical grade.g/min at 4 °C for 20 min. Finally, the pH of the upper layer solution was adjusted to 7.0 and stored at −20 °C for subsequent analysis. The simulated gastric and intestinal fluids were prepared according to the Chinese Pharmacopoeia and were prepared and maintained at 37 °C during experiments. Furthermore, the digestion samples were divided into two parts by ultrafiltration with a Molecular Weight Cut Off (MWCO) of 5 kDa membrane.The simulated gastrointestinal digestion was performed according to the method described by Linda and collaborators with some modifications . Briefly2, and 90% humidity for adaptive culture for 3–4 h. After the excess medium was removed, fresh medium was added.The B-lymphocytes were purchased and placed in a carbon dioxide incubator at 37 °C, 5% CO6 cells/mL. Normal saline was used as a control group, and the experimental groups were digested for samples less than 5 kDa and larger than 5 kDa after treatment. In this experiment, 3 parallel wells were used for each group and cultured in a 5% CO2 cell incubator at 37 °C for 48 h. The MTT solution was added to each well for 4 h before the end of the experiment and centrifuged for 5 min at a speed of 1000 r/min in a flat centrifuge 4 h later. The supernatant was discarded, and an appropriate amount of dimethyl sulfoxide was added to each well. After complete color development, the absorbance of each well was measured at 570 nm using a Bio-Rad 550 enzyme-linked immunosorbent assay. The B lymphocyte proliferation rate was calculated according to the following formula:A—The detected OD value; eA—OD value of the experimental group; cA—OD value of the control group.The Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to measure cell growth inhibition. For that, B lymphocytes of mice in the logarithmic growth stage were collected and added to a 96-well plate with the cell density adjusted to 5 × 105 seeded into a 96-well plate and 100 μL of RMPI-1640 were added, and then 50 μL of peptide fragments with the concentrations from 0 mM to 10 mM were added. Three parallel wells were set in each group. After culturing for 48 h under the condition of 37 °C and 5% CO2, experiments were conducted according to the operating procedures of the instructions of the TNF-α enzyme-linked immunosorbent assay kit [In total, 100 μL of B-lymphocytes with a cell density of 1 × 10Reverse phase high-performance liquid chromatography (RP-HPLC) was used to analyze the composition of peptides passed through the Caco-2 cell membrane. The detection conditions used in this experiment were listed in http://www.matrixscience.com/) to retrieve the uniprot database. Two-stage mass spectrometry was also performed for the identification of three peptide fragments .Mass spectrometry technology was mainly applied to the digestive peptidomics of protein. The mass spectrometric detection system is Q Exactive, equipped with nanoESI in the positive ion mode, for the search and identification of the characteristic peptide trypsin hydrolysate. The ionization source conditions were set as follows: capillary voltage, 3.5 kilovolts; source temperature, 275 °C; the collision gas is high purity nitrogen. The quantitation mode was performed in DDA mode. The other parameters are shown in 2 and relative humidity of 90% until the number of cells accounted for 80–90% of confluency [5 cells/mL, which was seeded on a 12-well Transwell plate added to the AP side of the 0.5 mL cell suspension, and the Basal lateral (BL) side by adding 1.5 mL of cell culture medium after one day after the exchange, Then, the fluid was changed every other day for 6 days, and the fluid was changed every day until the culture was completed. Cell membrane integrity and other indicators must be tested to successfully establish Caco-2 cell models. The main detection indicators were: (1) cell morphology observation; (2) the cell membrane compactness using fluorescent yellow test; (3) the detection of single-cell membrane resistance [Caco-2 cells were maintained in an incubator at 37 °C, with 5% COnfluency . After tsistance ; (4) alkPapp formula of fluorescence is as follows:0C—Initial concentration of fluorescent yellow (mg/mL); A—Membrane surface area (cm2); dQ/dt—Transport of fluorescent yellow per unit time; Papp—Apparent permeability coefficient.The The transport experiment was conducted with KHPIK to optimize the main conditions including concentration, contact time, pH, and transport direction, which could affect the transmembrane transport of bioactive peptides. 2 incubator for 30 min and followed by the removal of these compounds. The peptides were then added to the AP side, and Hank’s Balanced Salt Solution (HBSS) buffer was added to the BL side, and samples were collected after 2 h.After 0.5 mL of 10 mM Gly-Pro, 0.1 mM sodium deoxycholate and 0.025 mM phenylarsine oxide were added to different wells on the AP side, and the Transwell plates were incubated in a CO2 incubator for 30 min. After this incubation, the compounds were removed, the peptide was added to the AP side, and HBSS buffer was added to the BL side for 2 h.In total, 0.5 mL of 0.05 mM MK-571 sodium salt, 0.1 mM verapamil, and 10 mM sodium azide were added to the AP side hole of the Transwell plate and incubated in a COp < 0.05) via one-way analysis of variance (ANOVA).The amino acid sequence was analyzed by Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) sequential software and PEAKS 7.0 software. The data obtained was visualized using GraphPad Prism 6.01 software and analyzed using SPSS 22.0 software for significance analysis (http://mbpdb.nws.oregonstate.edu). S1-casein, and FFSD is derived from κ-casein. Furthermore, KHPIK and KHPIKH differed only in one amino acid, and KHPIK was also found to be active by highly credible peptide fragmentation pattern and matching in LC-MS/MS sequence software mascot 2.3.0. These hydrolysis-resistant peptides may exert immunoregulatory activity in vivo. Since polypeptides containing less than three amino acids cannot be detected by mass spectrometry, these kinds of peptides could not be identified in the casein-derived hydrolysate. Herein, four identified peptides including KHPIK, KHPIKH, RYPLGYL, FFSDK, and a triplet peptide YGG derived from κ-Casein with a strong proliferation effect on peripheral blood lymphocytes [The immunologic activity of the hydrolysate was explored. The results demonstrated that the casein hydrolysate and the permeate hydrolysate both promoted the proliferation of B-lymphocytes, indicating that the active immunologic mixture in casein hydrolysis was mainly less than 5 kDa . The majphocytes  were synp < 0.05). As the concentration was greater than 5 mM, the proliferation rate decreased because high concentrations might induce cell damage and peptide inhibition. The immune-active peptides detected in our study had a similar proliferation rate with the immune-active peptide GYPMYPLPR used in a previous study [The measurement of the effect of RYLGY on cell proliferation showed no significant difference with the control group, which indicated that the peptide did not promote the proliferation of B-lymphocytes. Therefore, RYLGY was selected as a control group for the immune activity experiment. The B-lymphocyte proliferation rates of the five synthesized short peptides were detected, and the results are shown in us study . Therefop < 0.05). Studies showed that other immunological active peptide fragments had similar ef-fects. For instance, the hazelnut hydrolysate containing the immunologically active peptide PEDEFR significantly inhibited the secretion of TNF-α [The synthesized five short peptides were treated with cell lines to detect the TNF-α secretion, and the results are shown in of TNF-α . The Caco-2 cell monolayer was evaluated at four aspects. The Caco-2 cells grew adherent to the wall after 21 days of culture and observed using an optical microscope A. The cePapp of fluorescence yellow was 6.07 × 10−7 cm/s, suggesting that the Caco-2 cell membrane was already very dense and could be used to understand the transport mechanism [2, proving that the cell lines model met the experimental conditions. The results in echanism . The trap < 0.05). Given the pH of human small intestinal cells is around 7.4, this can affect cellular physiological activity. The results also revealed that the transport quantity of KHPIK from the BL-AP side was significantly lower than that from the AP-BL side (p < 0.05), indicating that KHPIK is easier to be transported and absorbed from the top of intestinal cells to the basal side, so the transport direction was from AP to BL (AP-BL) side. However, a small amount of peptides transport from the BL-AP side suggests that the polypeptide transport may be related to efflux.The results in -BL side D , indicating that the carrier transport of peptides may not be the main transport mechanism for these peptides (p > 0.05). Therefore, the results indicate that endocytosis is not the main transport mechanism. However, when sodium deoxycholate was added, a significant increase in the transmembrane transport of these peptides was observed (p < 0.05). Overall, the results suggest that bypass diffusion is the main mechanism used to transport RYPLGYL, KHPIKH, KHPIK, FFSDK, and YGG in the intestinal epithelial cells.It has been described that polypeptides pass through cell membranes in the following ways: intercellular transport, passive transport, active carrier transport, endocytosis, and efflux . In passpeptides . PhenylaMultidrug resistance protein (MRP) and P-glycoprotein in Caco-2 cells are energy-dependent efflux transporters that use Adenosine triphosphate (ATP) as an energy source. P-glycoprotein on the AP side of the Caco-2 cells and the MRP2 in MRP can efflux cellular endogenous and exogenous substances to the AP side, resulting in less absorption. It has been stated that MK-571 sodium salt inhibits the efflux of MRP2, and on the other hand, verapamil can inhibit the efflux of P- glycoprotein. Furthermore, some studies have described that sodium azide is an inhibitor that can inhibit ATP production on the cell membrane. The inhibitors described above were used in this study to understand the underlying efflux mechanism of the immune-active peptides . p > 0.05), indicating that MRP2 and p glycoprotein did not interfere with the efflux of these three peptides. On the other hand, the concentration of RYPLGYL, KHPIKH, and KHPIK on the BL side increased significantly after adding sodium azide (p < 0.05), indicating that the transport of these three peptides depends on the consumption of ATP. Furthermore, the results also demonstrated that adding MK-571 sodium salt had no significant or differenct effects on the transport quantity of FFSDK compared with the blank group (p > 0.05). However, the transmembrane transport quantity of FFSDK significantly increased after adding verapamil and sodium azide (p < 0.05), indicating that FFSDK can use p-glycoprotein as a carrier and consume ATP to promote this transport. Moreover, p < 0.05), indicating that the YGG efflux during absorption is mainly mediated by P- glycoprotein. However, no significant differences were observed in the transport amount after adding MK-571 (p > 0.05). Additionally, the concentration of YGG in the BL side after adding sodium azide was slightly higher than in the blank group, suggesting that some ATP production was inhibited, impacting the transport amount.The results obtained in According to the RP-HPLC spectrum, there is degradation of peptides after passing through the cell membrane . FurtherIn vivo evaluation of bioactive peptides release and absorption in human beings are difficult to perform for the generation of great endogenous peptides. Hence, several in vitro digestion methods with simulating gastrointestinal tract were developed. Here, we identified 37 potential immune-active peptides by the simulated gastrointestinal digestion. Given the complexity of digestion and absorption process, it is difficult to trace the food-derived peptides in the gastrointestinal tract. Most studies developed the Caco-2 monolayer membrane to investigate the adsorption mechanisms.− 3 M, pH 7.4, transport in a direction from AP to BL, and transport time of 2 h. The absorption mechanisms of the five bioactive peptides in the Caco-2 cell model were all in the form of bypass transport. However, the transportation of RYPLGYL, KHPIK, and KHPIKH might be companied with efflux, which may require ATP to provide energy. The efflux of YGG during absorption was mainly mediated by P- glycoprotein. On the other hand, the FFSDK efflux was a transport process mediated by P- glycoprotein, and ATP was used as an energy source. Additionally, the LC-MS/MS results showed that some peptides  passed through the Caco-2 monolayer cell membrane and maintained their structural form. However, some were hydrolyzed into small-molecule polypeptides by the brush border enzyme system on the cell membrane. Rishika and collaborators studied the transport modes of three bioactive peptides derived from milk protein. They found that the transmembrane transport ratio of VLPVPQK was higher than TPFPG and RPGFSPFR since the heptapeptide was transported into the body as a carrier, and the primary mode of intestinal absorption of pentapeptide and nonapeptide was bypass transport [Relevant reports have shown that the polypeptides with amino acids in the range of 2–9 peptides containing more than 3 amino acids are more easily hydrolyzed, resulting in a low transport amount of the polypeptide by the cell membrane . The mairansport . Additioransport . Furtherransport . OverallAlthough the immunologically active peptides can completely cross the Caco-2 cell membrane, its interaction with related peptidases (such as DPP-IV) and transporters (P-glycoprotein) during transport remains to be further verified. In fact, the effects of other food substrates including starch, polysaccharide, lipid etc. on digestion and absorption should also be investigated for real food ingestion.−7 cm/s, 2.60 × 10−7 cm/s, 3.22 × 10−7 cm/s, 4.91 × 10−7 cm/s, and 5.12% × 10−7 cm/s. The LC-MS/MS identified the permeant fluid and found that one part of this peptide was completely absorbed by intestinal epithelial cells, and the other was hydrolyzed. These suggest that these immunologically active polypeptides could be absorbed and may play a role in addition to its enteric cavity. It is expected that this conclusion can be improved through animal experiments.Casein hydrolysis presented the immune activity through the simulated gastrointestinal method, and these results were identified by the LC-MS/MS. The five peptides in the study, RYPLGYL, KHPIKH, KHPIK, FFSDK, and YGG, were synthesized, and the results proved that they had immunological activity by promoting the proliferation rate of B lymphocytes and reducing the secretion of TNF-α. Furthermore, the transport mechanism of these five peptides occurred through bypass on the Caco-2 monolayer cell model. On the other hand, the Papp values of RYPLGYL, KHPIKH, KHPIK, FFSDK, and YGG within 2 h were 1.61 × 10


Mrakia gelida MGH-2 has been reported from Surikogi Ike in the Skarvsnes ice-free area, East Antarctica. Here, we report on the high-quality genome sequence of the Mrakia gelida MGH-2 strain analyzed by PacBio Sequel and HiSeq 2500 instruments.Basidiomycetous yeast  Mrakia gelida MGH-2 was isolated from lake sediment of Surikogi Ike in the Skarvsnes ice-free area, East Antarctica, by the 48th Japanese Antarctic Research Expedition (JARE-48) (Basidiomycetous yeast JARE-48) . This stg for 5 min at 4°C. The cell pellets were washed with sterile distilled water and were precipitated by centrifugation again. The cell pellets were dried by a vacuum freeze dryer. Then, the lyophilized cells were powdered in a mortar and used for DNA extraction. The genomic DNA of MGH-2 was extracted and purified using a NucleoBond AXG100 column with a Nucleo buffer set III (TaKaRa Bio) according to the manufacturer’s protocol. Concentration and purification of the genomic DNA were determined by NanoDrop (Thermo Scientific) and the Qubit double-stranded DNA (dsDNA) broad-range (BR) assay kits (Thermo Scientific). Then, the genomic DNA was divided into two parts, namely, one for the PacBio Sequel platform and the other for the HiSeq 2500 platform  for use in sequencing. For the PacBio Sequel platform, a single-molecule real-time (SMRT) sequence library was constructed using a SMRTbell express template prep kit v1.0  according to the manufacturer’s protocol. Two sequencing libraries were size selected using the BluePippin system  with a minimum fragment length cutoff of 20 kbp and 30 kbp. Each library was sequenced on one SMRT cell with binding kit 1.0/sequencing kit 1.2.1 and binding kit 2.0/sequencing kit 2.0, respectively. The run time for the movies was 360 min and 600 min, yielding a total of 1,133,939 polymerase reads. In this study, default parameters were used except where otherwise noted. In the PacBio Sequel sequencing, read quality control was performed prior to assembly by measuring the average length and concordance of spike-in controls and the distribution of read lengths in the sample. PacBio reads were assembled using the FALCON-Unzip assembler program v0.7 (The MGH-2 strain culture and DNA extraction methods are the same as those we reported previously , 4. BrieM. gelida MGH-2 is shown in A summary of the genomic analysis results of the M. gelida MGH-2 will be useful in the search for new bioactive substances and new pharmaceutical raw materials.Antarctica is one of the last frontiers for bioprospecting, along with deep sea and space. We believe that genomic information on PRJDB13796 with the accession no. BRON00000000.1. The raw reads are available under the DRA accession no. DRA014407.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under BioProject no.


Significantly lower plasma concentrations of butyric, isobutyric-, and isovaleric acid were detected in AN as well as AN-REC compared with CTRL. We also show significant correlations between plasma concentrations of SCFA and BMI. These results encourage studies evaluating whether interventions directed toward altering gut microbiota and SCFA could support weight restoration in AN.Anorexia nervosa (AN) is one of the most lethal psychiatric disorders. To date, we lack adequate knowledge about the (neuro)biological mechanisms of this disorder to inform evidence-based pharmacological treatment. Gut dysbiosis is a trending topic in mental health, including AN. Communication between the gut microbiota and the brain is partly mediated by metabolites produced by the gut microbiota such as short-chain fatty acids (SCFA). Previous research has suggested a role of SCFA in weight regulation . Moreover, fecal SCFA concentrations are reported to be altered in active AN. However, data concerning SCFA concentrations in individuals who have recovered from AN are limited. In the present study, we analyzed and compared the plasma concentrations of seven SCFA  in females with active AN ( Up to 4% of females and 0.3% of males are estimated to be diagnosed with the psychiatric disorder anorexia nervosa (AN) during their lifetime , a disorSeveral studies have documented a dysbiosis of gut microbiota in individuals with AN who experience drastically altered diets and prolonged low energy intake ,12,13,14Roseburia, as well as an increase of mucine-degrading species such as Akkermansia, may represent hallmarks of the gut microbiota alterations in AN. Interestingly, Akkermansia supplementation has based on its beneficial effects on health and metabolism been suggested as an intervention for obesity [The gut communicates with other organs including the brain  via metabolites produced by the microbiota. Upon microbial fermentation of carbohydrates, such as dietary fibers and resistant starch, short-chain fatty acids (SCFAs) are produced. Lower fecal concentrations of the SCFAs butyrate and propionate, and occasionally acetate, have previously been reported in individuals with active AN ,10,21,22 obesity . Moreove obesity . Lastly, obesity ,13,14,22(n = 109). For AN-REC, the inclusion criteria were a history of DSM-IV AN diagnosis followed by weight restoration (BMI > 20 kg/m2), plus no eating disorder behaviors for at least a year (n = 108). One year was chosen based on the recovery literature since it is the most common minimum symptom-free duration required to be considered recovered, and ensures to some extent, that relapse is less likely [n = 110). Stratified analyses of the two AN groups were carried out exploring subtypes; AN with binge  as defined by episode(s) of binge eating with loss of control, while complete absence of such episodes was defined as AN without binge eating . We also performed a stratified analysis of active AN, as well as AN-REC, with documented episodes of laxative use  compared with without laxative use , as well as with documented self-induced vomiting  compared to without vomiting . See This cross-sectional study included 327 females all identified from the Swedish sample of the Anorexia Nervosa Genetics Initiative (ANGI-SE). Details on the recruitment procedure have been described by Thornton et al. . The incBlood was collected using EDTA tubes at a hospital or a lab near the participant, sent to Karolinska Institutet Biobank with overnight mail, and processed upon arrival. After centrifugation, plasma samples were stored at −80 °C pending analysis at the Chalmers University of Technology.g for 5 min and the supernatant was moved to a new tube. The sample was made up to 1 mL by 10% methanol in water and again centrifuged at 15,000× g for 5 min. In total 100 μL of the derivatized (12C) sample was mixed with 100 μL of labeled (13C) internal standard. Samples were analyzed by a 6500+ QTRAP triple-quadrupole mass spectrometer  which was equipped with an APCI source and operated in the negative-ion mode. A Phenomenex Kinetix Core-Shell C18  UPLC column with SecurityGuard ULTRA Cartridges (C18 2.1 mm ID)  was used for separation of the analytes. The column was backflushed for 60 min between each batch to ensure good chromatographic separation. LC-MS grade water (100% solvent A) and acetonitrile (100% solvent B) were the mobile phases for gradient elution. The column flow rate was 0.4 mL/min and the column temperature was 40 °C, the autosampler was kept at 5 °C. The gradient started at 0.5% B (held for 3 min), 2.5% B ramping linearly to 17% B at 6 min, then to 45% B at 10 min, and 55% B at 13 min. Followed by a flush (100% B) and recondition (0.5% B), a total runtime of 15 min. The multiple reaction monitoring (MRM) transitions were optimized for the analytes one by one by direct infusion of the derivatives containing 50 mM of each fatty acid. The Q1/Q3 pairs were used in the MRM scan mode to optimize the collision energies for each analyte, and the two most sensitive pairs per analyte were used for the subsequent analyses. The retention time window for the scheduled MRM was 1 min for each analyte. For the two MRM transitions per analyte, the Q1/Q3 pair that showed the higher sensitivity was selected as the MRM transition for quantitation. The other transition acted as a qualifier for verification of the identity of the compound. Linear, eight-point calibration curves were prepared for each reference compound and used for quantification. The intra-and inter-batch variations were calculated based on the inclusion of three different QC samples with different concentrations in triplicates in each batch across seven batches. The mean intra-batch variation was between 3–11% for all SCFA except valeric acid (>15%), for which the inter-batch variation also was high (>40%), and subsequently was not included in further analyses. Caproic acid was also excluded since unpublished data from our lab on another cohort of plasma show a low run-rerun correlation for this low abundant SCFA. The inter-batch variation was controlled for by normalizing the sample values with the QC values. The normalization factor for each analyte per batch was calculated by the mean of QC values of the individual batch/mean of the total QC values from all seven batches. The statistical analyses were performed on normalized data.Nine SCFAs  were analyzed in EDTA plasma by liquid chromatography-mass spectrometry (LC-MS) according to a method described previously  with somDemographic and clinical characteristics of AN, AN-REC, and CTRL groups were analyzed using descriptive statistics. Group differences in plasma concentrations of the seven SCFAs  in AN, AN-REC, and CTRL were analyzed using the Kruskal-Wallis test since the plasma concentrations of analytes were not normally distributed, followed by posthoc Dunn’s test with Bonferroni correction to evaluate pairwise comparisons. Group differences in plasma concentrations of SCFA in AN-B vs. AN-noB, AN-REC-B vs. AN-REC-noB, AN-LAX vs. AN-noLAX, AN-REC-LAX vs. AN-REC-noLAX, AN-VOM vs. AN-noVOM, as well as AN-REC-VOM vs AN-REC-noVOM were tested using the nonparametric Mann–Whitney U test.p-values to account for the three independent tests, and an alpha level of 0.017 was therefore considered statistically significant. All statistical analyses were performed using R programming language version 4.1.0 (including package emmeans). Graphs were made using the ggplot2 package from R.Associations between SCFA concentrations and age, BMI, as well as years since AN onset, were assessed using linear regression models. If a significant correlation between SCFA levels and age was found, we would adjust for age in further analyses for this SCFA. Spearman correlation revealed that the plasma concentrations of five out of seven SCFA correlate with each other . We thusp = 1.69 × 10−30), and AN vs. CTRL (p = 1.67 × 10−44), minimum BMI AN vs. AN-REC (p = 8.02 × 10−19), as well as length of amenorrhea AN vs. AN-REC (p = 3.29 × 10−6). Psychiatric comorbidities of the study participants are summarized in The demographic and clinical characteristics of the study population are summarized in We observed no effect of age at sampling on normalized plasma concentrations of any of the seven SCFAs analyzed, thus further analyses were not adjusted for age. Analyses of group differences showed that plasma concentrations of butyric-, isobutyric-and isovaleric acid were significantly lower not only in AN compared with CTRL, but also in AN-REC compared with CTRL .p = 0.004), as well as between AN and AN-REC (p = 0.005), for propionic acid. The same was seen for succinic acid, AN vs CTRL (p = 0.005) and AN vs AN-REC (p = 0.007). No significant differences were found between the AN-REC and CTRL groups with respect to correlations between BMI and SCFA concentrations  . MoreoveThe potential causality between deviant SCFA plasma concentrations and AN cannot be elucidated with the present design. However, preclinical and clinical studies, including fecal transplantation, support the role of the gut microbiome and SCFA in weight regulation ,16,19,20anx/anx mouse [Other routes by which SCFA could influence weight and food intake regulation are via hypothalamic inflammation and microglia. SCFAs—in particular butyrate—here shown to be lower in AN and AN-REC plasma, are crucial for microglia maturation and activation. These brain cells appear to require continuous stimulation from the gut microbiota to remain mature ,50. Micrnx mouse . Similarnx mouse ,54. Recenx mouse . In addinx mouse ,57,58, wnx mouse ,60. ThusThe main limitation of our study is the cross-sectional design, which precludes inferences about causality. Another limitation is the blood sampling procedure, as blood samples were sent via overnight mail before being processed. Even if this may have influenced the absolute SCFA concentration , it is uFuture research should evaluate plasma concentrations of SCFA in AN-REC with a longer defined time since recovery. The optimal design would be a longitudinal study that followed individuals over the course of treatment through recovery that measured SCFA concentrations in plasma and feces combined with dietary records to decipher the effect of nutritional recovery post AN on the gut microbiota and its metabolites. Lastly, it would also be valuable to further explore the effects of SCFA on glial cells and neurons of interest for the AN pathology .Here, we report significantly lower plasma concentrations of butyric-, isobutyric-, and isovaleric acid in individuals with active AN—as well as in individuals who had recovered from AN for at least one year—compared with healthy controls. This combined with the knowledge that SCFA can intervene with several food intake and body weight regulating mechanisms, suggests an opportunity to test the augmentation of standard treatment with these specific metabolites as a potential route to support, enhance, and maintain weight recovery in AN . Althoug


Nanocomposite hydrogels have attracted researchers’ attention in recent years to achieve superior performances in a variety of materials applications. In this work, we describe the outcome of three different strategies to combine a self-assembling tripeptide and carbon nano-onions (CNOs), through covalent and non-covalent approaches, into supramolecular and nanostructured hydrogels. Importantly, the tripeptide coated the nano-onions and extended their aqueous dispersions’ stability by several hours. Furthermore, CNOs could be loaded in the tripeptide hydrogels at the highest level ever reported for nanocarbons, indicating high compatibility between the components. The materials were formed in phosphate-buffered solutions, thus paving the way for biological applications, and were characterized by several spectroscopic, microscopic, thermogravimetric, and rheological techniques. In vitro experiments demonstrated excellent cytocompatibility. Nanocomposites are multiphase materials that incorporate structures that, by definition, have dimensions in the nanoscale. Recently, we have witnessed the rise of nanocomposites, thanks to their great versatility for various applications . The incSoft matter, such as hydrogels, can benefit from the inclusion of CNMs to gain new mechanical, thermal, and electronic properties ,41,42,43Despite the increasing interest in the emerging area of nanocomposites incorporating both CNMs and self-assembling short peptides , to the In this work, we explored three functionalization routes to combine CNOs with a self-assembling short peptide into hydrogels. One key aspect to bear in mind is the dispersibility of the CNM in the solvent of choice, which is required to ensure the final nanocomposite’s best performance. With the choice of solvent in consideration, the CNO-peptide hydrogels were prepared in phosphate buffer. These solutions are physiologically compatible and could pave the way for future applications of CNO-peptide hydrogels as nanocomposite biomaterials. Indeed, in previous studies, CNOs displayed an excellent in vitro ,61 and iAll solvents and reagents were acquired from Merck  and used as received unless otherwise specified. High-purity Milli-Q water with a minimum resistivity of 18.2 MΩ·cm @ 25 °C was produced from a Milli-Q Academic System  and employed to prepare all solutions and buffers. The microwave (MW)-assisted synthesis was carried out in the Microwave reactor Discover SP–CEM Corporation . The sonicator used was the Branson Ultrasonic 3800 cleaning bath .–1 up to 800 °C.Thermogravimetric analyses (TGA) were carried out on a TGA5500  under a nitrogen and an air atmosphere. For each sample, 1 mg of material was analyzed by equilibrating at 100 °C for 20 min and then applying a heating ramp of 10 °C min−1 resolution, 240 scans, with an ATR-FTIR Affinity-1S . Every sample was analyzed at least three times.Attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) spectra were acquired at 4 cm−1 resolution. Raman mapping was carried out based on the intensity ratio of the signals at 1351 and 1002 cm−1.Samples were prepared on microscopy glass slides and dried overnight. Raman spectra were acquired on five different spots per sample with an inVia Renishaw microspectrometer  equipped with laser at 532 nm (25 mW), with 1 accumulation, 1 cmTransmission electron microscopy (TEM) was carried out on a JEM 2100  at 100 kV. Samples were transferred onto TEM carbon grids previously exposed to UV-Ozone Procleaner Plus for 6 min and dried in vacuo. The grid samples were stained with potassium phosphotungstate (2% pH 7.2). Electron micrographs were analyzed with FIJI free software (ImageJ 2).Rheological analyses were performed on a Malvern Kinexus Ultra Plus Rheometer  using a 20 mm stainless-steel, parallel-plate geometry with a 1 mm gap at a fixed temperature of 25 °C using a Peltier temperature controller . The hydrogels were formed directly on the rheometer plate by mixing 200 μL of the CNOs dispersion in the peptide solution in the alkaline buffer and the same amount of the acidic buffer. Time sweeps were performed at 1 Pa and 1 Hz for 1 h. Frequency sweeps were recorded at 2 Pa from 0.1 to 10 Hz. Stress sweeps were performed from 1 Pa up to gel failure at 1 Hz.All reverse-phase HPLC purifications were carried out on the 1260 Agilent Infinity system equipped with a preparative gradient pump (G1311B), semipreparative C-18 column  and the photodiode array detector (G1315C) set at 214 and 254 nm. All LC-MS analyses were carried out on the 6120 Agilent Infinity system that consists of the 1260 described above connected to the ESI-MS detector, single quadrupole.13C and 400 MHz for 1H nuclei. Samples were dissolved in deuterated solvents as indicated in the NMR spectra were acquired on a Varian 400 MHz  at 101 MHz for ® Molto from Carbodeon Ltd., Vantaa, Finland; 5 nm average particle size) was thermally annealed at 1650 °C under a positive pressure of helium to produce small p-CNOs . The procedure was carried out as previously reported [Detonation nanodiamond powder , excess nitric acid was removed through centrifugation, removal of the supernatant, and redispersion of the material in deionized water. The oxi-CNOs were then filtered off on a Millipore membrane  and sequentially washed with water, methanol, and acetone. After drying overnight at RT, the oxi-CNOs were recovered as black powder .N-hydroxysuccinimide/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (NHS/EDC·HCl) mediated amidation coupling in dry N,N-dimethylformamide (DMF), with 4-dimethylaminopyridine (DMAP) added as a catalyst. The coupling reaction was carried out with stirring under reflux at 140 °C for 3 days. After cooling to RT, the amino-PEG-CNOs were filtered off on a Millipore membrane  and sequentially washed with DMF, water, methanol, and acetone. After drying overnight at RT, the amino-PEG-CNOs were recovered as black powder.The amino-terminated PEGylated CNOs (amino-PEG-CNOs) were prepared as previously described . PEGylatThe tripeptide L-Leu-D-Phe-D-Phe (Lff) was synthesized by solid phase according to published procedures, using 2-chlorotrityl chloride resin . Lff wasN-protection strategy to form Fmoc-Lff was carried out as per published procedures [The Lff Fmoc ocedures . In contTo prepare the amino-terminated PEGylated Lff, Fmoc-Lff was prepared as described above—however, prior to cleaving the product off the resin, leucine was deprotected and the Fmoc-8-amino-3,6-dioxaoctaoic acid  PEG linker was added by treating it just like an amino acid, and then likewise it was Fmoc-deprotected and the product was cleaved off the resin through acidic hydrolysis. Amino-PEG-Lff was purified by dissolving it in MeCN/water 30:70 (with 0.05% of TFA in both solvents) and then the mixture was filtered with a 0.45 μm PTFE filter and injected in the HPLC using the system described above  and the following method: t = 0–2 min, 20% MeCN; t = 152 min, 50% MeCN; t = 153 min, 95% MeCN; t = 158 min 95% MeCN (Rt = 53 min).N,N′-diisopropylcarbodiimide (DIC) (3 eq) and Fmoc-Lff (3 eq) in DMF (2 mL) was added, and the final mixture was kept under vigorous magnetic stirring for 24 h at RT. The crude was filtered on a Millipore membrane  and washed with DMF three times, twice with DCM, water and methanol, and then with diethyl ether. The product was left to dry overnight on the filter. For the MW-assisted coupling, amino-PEG-CNOs (10 mg) were placed in a MW reactor vessel and dispersed in DMF (2 mL) by sonication (30 min). A solution of Oxyma Pure B (3 eq), DIC (3 eq), and Fmoc-Lff (3 eq) in 1 mL of DMF was added, and the final mixture was put in the MW reactor for 15 min at 80 °C, with a power of 150 W. After that time, the crude was filtered on a Millipore membrane  and washed as indicated above. The deprotection was performed by stirring the coupling product for 30 min at RT in 20% piperidine in DMF (10 mL). The final product was filtered, washed, and dried as indicated above.Amino-PEG-CNOs (15 mg) were placed in a round-bottom flask and sonicated for 30 min in DMF (13 mL). Next, a solution of Oxyma Pure B (3 eq), Oxi-CNOs (10 mg) were placed in a round-bottom flask and sonicated for 30 min in DMF (6 mL). A solution of Oxyma Pure B (5 eq) and DIC (3 eq) in DMF (2 mL) was added, and the resulting mixture was kept under vigorous magnetic stirring for 16 h at RT. Next, a solution of amino-PEG-Lff (5 eq) in DMF (2 mL) was added, and the final mixture was kept under vigorous magnetic stirring for 24 h at RT. The crude was filtered on a Millipore membrane  and washed with DMF three times, DCM, water and methanol two times for each solvent, and the last time with diethyl ether, and the product was left to dry overnight at RT.1.0, 2.0, or 4.0 mg of oxi-CNOs  or 2.0 mg of Lff-PEG-CNOs  were added to 1.0 mL of a solution containing 5.0 mg of Lff in alkaline sodium phosphate  and sonicated for 15 min. Then, an equal volume of a mildly acidic sodium phosphate buffer  was added to reach a pH of 7.4 and trigger the peptide self-assembly and hydrogelation.n = 3).Oxi-CNOs and Lff-PEG-CNOs were dispersed in the gel-precursor solutions with or without Lff tripeptide at the concentration of 0.2 μg/mL and placed inside the cuvettes for the measurement of ζ-potential in a Malvern Zetasizer Nano . Measurements were performed at 25 °C and average values were calculated with Excel  with oxi-CNOs  and Lff tripeptide were prepared as described in Non-covalent hydrogels (70 μL) were prepared with  or without oxi-CNOs as described in 2 for 24 h, by handling the slides according to the manufacturer’s instructions. Acridine orange (5 μL/well of a 20 μM solution in 50 mM PBS) and propidium iodide (5 μL/well of a 30 μM solution in 50 mM PBS) were added in each well. Cells were imaged on a Leica microscope  with green and red filters and a 10× objective. Each condition was repeated 3 times.The live/dead assay was performed according to a modified protocol . Gels  were added, and the microplate was incubated for 4 h in a humidified atmosphere . Next, 100 μL of the solubilization solution for formazan crystals (4 mM HCl + 0.1% IGEPAL in isopropanol) was added to each well, and the microplate was kept at room temperature under shaking  for 30 min. To solubilize the gels, 7 μL methanol were added to each well, and samples were pipetted up and down to obtain a homogeneous solution. After visual inspection for complete solubilization of the purple formazan crystals, the absorbance was read at 570 nm with a reference wavelength of 690 nm (light scattering and oxi-CNOs absorbance) using a microplate reader . Each condition was repeated in 6 replicas and statistical analysis performed in Excel as described in Hydrogels (50 μL) were prepared without or with oxi-CNOs  as described in p < 0.05 was used as reference value for statistical significance.Statistical analysis was performed using the t test formula in Excel over 6 replicas for each tested condition. Non-covalent approach ;C-terminal covalent approach ;N-terminal covalent approach .The tripeptide L-Leu-D-Phe-D-Phe (Lff) was recently reported for its self-assembling ability in phosphate-buffered solutions . The tri1H-NMR, and 13C-NMR  or through the intercalation of the bound peptide on the CNO surface within the stacks of free peptide (routes 2 and 3). For the three strategies, the Lff tripeptide and its derivatives were synthesized by solid phase, purified, and characterized by LC-MS,  13C-NMR .2 C atoms to sp3 with the consequent introduction of defects in the structure, the CNOs decomposition temperature decreased from 688 °C (p-CNOs) to 653 °C (oxi-CNOs) and 649 °C (amino-PEG-CNOs). Pristine CNOs (p-CNOs) were firstly oxidized using an established procedure to introduce COOH groups on their surface and render them more easily dispersible in aqueous conditions. Next, they were functionalized with a PEG linker to favor peptide covalent attachment . Charact−1 that originated from the stretching modes of sp2 carbon atoms. As expected, the oxi-CNO spectra presented two signals at 1739 and 1209 cm−1 from C=O and C-OH stretching modes, respectively. These same signals were found in the spectra of amino-PEG-CNOs, indicating that not all surface carboxylic acid groups were PEGylated. Free carboxylic groups on the CNO surface are desirable to maintain sufficient hydrophilicity for the material to be well dispersed in phosphate buffer. Nevertheless, the successful functionalization was confirmed by the fact that linker signals were predominant in the spectra of amino-PEG-CNOs, in particular those at 3310 cm−1 and 1658 cm−1 assigned to the N-H and C=O stretching of amide groups, respectively, together with signals arising from the linker chain . Likewise, Raman spectra  phosphate solution. Oxidation of p-CNOs is an established strategy to render them more hydrophilic and, thus, dispersible in aqueous solutions. Indeed, oxi-CNOs dispersed well; however, after 20 min, they started to deposit at the bottom of the vial . Conversreported , indicatreported , nanocarn = 50) diameter that interacted on their surface with individual CNOs that appeared to be well-dispersed within the matrix (Transmission electron microscopy (TEM) imaging revealed a network of highly homogeneous fibrils of 21 ± 2 nm  and the signal of the Phe rings at 1002 cm−1, showed that oxi-CNOs coated the peptide fiber, while their presence in the internal part was modest.A Raman mapping of a hydrogel fiber b, based Oscillatory rheological kinetic analysis a revealeC-terminus of Lff was too hindered due to the bulky Phe sidechain to allow its coupling to the amino-PEG-CNOs in high yields. Therefore, the peptide coupling was attempted via the N-terminus, as described in the following section.The coupling of the Lff peptide via the C-terminus to the amino-PEG-CNOs was attempted through several approaches, using different coupling agents, solvents, and also with the assistance of the MW-reactor. The best results were obtained with Oxyma Pure B and DIC as coupling agents in DMF; however, the thermogravimetric analysis revealed a negligible increase in the weight loss relative to the amino-PEG-CNOs precursor (ca. 1% wt.) that was not significantly different from the range of experimental error. It is possible that the D/IG value of 1.19 ± 0.83 was unsurprisingly similar to that of oxi-CNOs since the coupling reaction did not introduce new defects on the CNO surface. While the degree of functionalization is low, it is suitable for the purpose of this work—residual carboxylic groups on the CNOs surface are necessary to ensure their dispersibility in aqueous environments, as confirmed with the gel-precursor solution  phosphate solutions a, which Oscillatory rheology revealed similar gelation kinetics relative to the nanocomposite supramolecular hydrogel obtained through the non-covalent approach a. An incTEM micrographs revealed that the gel obtained through the covalent approach displayed not only a dense network of fibrils—analogously to the non-covalent gel —but alsoThe data above indicated that the nanocomposites formed with the non-covalent approach were more homogeneous, without aggregates, and with better coating of the oxi-CNOs by the peptide, relative to those formed through the covalent approach. The colloidal stability of the CNOs in the gel-precursor solutions was further probed by zeta (ζ) potential measurements to confirm these findings. This parameter is defined as the potential at the slipping plane, which is related to the surface charge of the CNOs and the chemical composition of the local environment. It provides an accurate estimation of the aggregation tendency of the nanoparticles, that is significantly reduced by electrostatic-charge repulsion for values that exceed +30 or −30 mV, and that provide moderate colloidal stability . Accordip < 0.005), and increased with higher loadings of oxi-CNOs, indicating a slight increase in hydrophobicity.In light of these findings, the non-covalent approach was thus selected for subsequent cytocompatibility studies. Firstly, the stability of the nanocomposite hydrogels was tested at physiological conditions, i.e., in phosphate-buffered-saline (PBS) solutions at 37 °C, with mild agitation. Different oxi-CNO loading levels were probed, ranging from 10 μg/mL to 1 mg/mL. Lower levels were not tested due to detection limits of the experimental setup, which used visible-light absorbance to monitor oxi-CNOs release from the biomaterial scaffold. Over the course of 24 h no release of oxi-CNOs was noted for the hydrogels loaded with 10 or 100 μg/mL, and <1% oxi-CNOs were released from the nanocomposite with the highest loading see . The watHaving established the biomaterials’ stability, their cytocompatibility was probed in vitro. The hydrogels were prepared with three different loading levels of oxi-CNOs that could ensure sufficient optical transparency for light microscopy, i.e., 0.1, 1.0, and 10 μg/mL. Peptide hydrogels without oxi-CNOs served as controls for the 3D scaffold. Fibroblast cells seeded on the hydrogels adhered to, and proliferated well into, all the biomaterials, with no significant difference between the tested conditions . FurtherMetabolic assays were performed to obtain another quantitative measure of cell proliferation in the presence of the nanocomposite hydrogels . To thisw/w relative to the peptide did not interfere with peptide self-organization into fibrils, surpassing all the other CNMs tested thus far [In conclusion, this work demonstrated that oxi-CNOs and the self-assembling tripeptide Lff are highly compatible with attaining nanocomposite supramolecular hydrogels at physiological conditions  for biological use. The presence of oxi-CNOs also at the remarkably high loading of 80% thus far . FurtherThe nanocomposite supramolecular hydrogels obtained through the non-covalent approach, i.e., by simply mixing the oxi-CNOs with the Lff tripeptide, were thoroughly characterized by spectroscopic, microscopic, and thermogravimetric analyses and demonstrated interactions between the two components as well as the absence of oxi-CNO aggregates within the hydrogel matrix that ensured a homogenous dispersion of the nanocarbon. These hydrogels displayed good hydrophilicity as required for biomaterial scaffolds, with contact angles in the range of 39–44°, depending on the loading level of oxi-CNOs. These biomaterials also displayed excellent stability at physiological conditions, with negligible release of oxi-CNOs within 24 h, and excellent cytocompatibility in vitro.The covalent binding of the Lff tripeptide with the oxi-CNOs was also attempted, but very low levels of peptide loading were obtained, and the material displayed improved rheological properties at the expense of CNO aggregation. Indeed, anchoring of the self-assembling tripeptide on the CNOs had a two-fold outcome: on the one hand, it acted as a reticulating agent by providing connection points between fibrils, but on the other hand, it also caused undesirable aggregation leading to a heterogeneous material. All in all, the non-covalent approach was the best strategy, especially considering its simplicity. This work opened the way to developing nanocomposite hydrogel biomaterials based on simple tripeptides and oxi-CNOs for advanced applications, such as targeted drug delivery and bioimaging.


The performance of the fPOC was evaluated with 12 SARS-CoV-2 clinical throat swab samples that included seven positive and five negative samples, as confirmed by reverse transcription–polymerase chain reaction. The fPOC showed 100% agreement with the commercial thermocycler. This simple design of the fPOC system demonstrates the potential to greatly enhance the practical applicability to develop a totally integrated point-of-care system for rapid on-site screening of the SARS-CoV-2 virus in the management of the pandemic.The COVID-19 pandemic emphasized the importance of rapid, portable, and on-site testing technologies necessary for resource-limited settings for effective testing and screening to reduce spreading of the infection. Realizing this, we developed a fluorescence-based point-of-care (fPOC) detection system with real-time reverse transcriptase loop-mediated isothermal amplification for rapid and quantitative detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The system is built based on the Arduino platform compatible with commercially available open-source hardware–software and off-the-shelf electronic components. The fPOC system comprises of three main components: 1) an instrument with integrated heaters, 2) optical detection components, and 3) an injection-molded polymeric cartridge. The system was tested and experimentally proved to be able to use for fast detection of the SARS-CoV-2 virus in real-time in less than 30 min. Preliminary results of testing the performance of the fPOC revealed that the fPOC could detect the SARS-CoV-2 virus at a limit of detection (LOD The concept of point-of-care (POC) testing originated in the 15th century ; howeverhttps://covid19.who.int/). The pandemic not only demonstrated a severe effect on health but also caused social turmoil and economic disruption . Researchers showed that the disease spreads easily through contact, droplets, airborne, fomite, fecal–oral, mother-to-child, and animal-to-human (https://www.who.int/news-room/commentaries/detail/transmission-of-sars-cov-2-implications-for-infection-prevention-precautions). Preventing the transmission of the disease will remarkably reduce the negative effects on the health, economy, and society.The COVID-19 pandemic is caused by a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). According to the World Health Organization (WHO), by the 20th of February 2022, the COVID-19 pandemic affected 220 countries, areas, and territories with more than 492 million confirmed cases and more than 6.1 million deaths (https://apps.who.int/iris/handle/10665/340072). Next, reverse transcriptase polymerase chain reaction (rRT-PCR) and later on antigen-based lateral flow tests were implemented as officially approved methods to detect SARS-CoV-2 in the clinical samples. However, the rRT-PCR is a time-consuming assay that requires sophisticated laboratory facilities, well-trained personnel, and the performance may be influenced by inhibitory effect . Another major drawback of rRT-PCR is its high cost/assay that was beyond the reach of resource-limited countries . For example, major lateral flow based quick testing kits such as Panbio™ COVID-19 Ag Rapid Test Device from Abbott  claim >99% positive agreement in the clinical sensitivity with the samples demonstrating Ct values ≤ 33  with a limit of detection (LOD) at ∼0.25 TCID50/μL of SARS-CoV-2 with a clinical sensitivity of 91.4%. All of these have become bottleneck in the rapid surveillance of SARS-CoV-2 and effective management of the pandemic. Loop-Mediated Isothermal Amplification (LAMP) has been demonstrated as a powerful alternative to overcome the drawback of PCR  and off-the-shelf electronic components (https://dk.farnell.com/) https://dk.rs-online.com/). , in the Supplement Information, shows a list of components used in the system and suppliers.Light sources: Five different blue light emitting diodes (LED) demonstrating a center emitting wavelength at 470 nm, with different viewing angle and maximum luminous intensity, were used as the light sources to optimize the excitation light .Filters: Two sets of excitation (EX) and emission (EM) filters, 1) Omega filters 465DF10–465 nm and 510DF10–510 nm with bandpass of 10 nm; https://www.omegafilters.com/optical-filters/bandpass and 2) Optolong filters 480 nm and 530 nm with bandpass of 30 nm, http://www.optolong.com/) were used to collect and extract the required excitation and emission fluorescence light wavelengths, respectively.Heaters: Two heating elements from Keenovo silicone heaters (www.keenovo.com), 60 mm × 60 mm in size, at power of 50 W @ 30 V, were used to build the heater system. The heating elements were stalked as such that the polymeric cartridge is sandwiched between top and bottom heating elements. Next, the top heater demonstrated custom made holes Ø of 5 mm under EX and EM filters in order to let the excitation light go through the sample and fluorescence emission light back to the detector.https://www.parallax.com/package/plx-daq).Parallax Data Acquisition tool (PLX-DAQ), an open access freely downloadable software) run on an external computer, was used to receive and process the data sent from the system , 1.67 µM of SYTO-9 (Invitrogen), sterilized water, and 6 μL template.LAMP primer set targeting gene N (nucleocapsid phosphoprotein) of SARS-CoV-2 was selected for this study . The rRTin-house developed turbidity-based POC device - the PATHPOD system  and used as positive control.5 dilution of plasmid control ranging from 48,000 to three copies per reaction was analyzed in three replicates. Negative oral swab sample without any target was tested in replicates of 3 as the negative control (NC). The threshold time (Tt) was recorded for NC that was at 32.76 Tt. The lowest concentration (76.8 copies/reaction) that gave positive results in all three repetitions was considered LOD of the system with 100% confidence level. Next, target concentration at 15.36 gave partial positive results, with two out of four reactions showing positive results, and that was considered as LOD50%. LOQ was estimated by adding the average Tt value at LOD50% (21.32) and the standard deviation (2.32) of the lowest concentration tested with a 100% confidence level.To estimate the LOD and limit of quantification (LOQ), a logThroat swab samples from voluntary individuals were collected at Hvidovre hospital, Denmark during the COVID-19 pandemic. The RNA from the samples were extracted and purified by MagNA pure 96 DNA and viral NA small volume kit , following the instruction of the manufacturer. Six µL of the extracted solution were used as template in the rRT-LAMP reaction.The fPOC working principle  is basedThe well is heated rapidly and maintained at the reaction temperature (60°C–65°C) by the two heaters: bottom and transparent top heater, respectively . The tarin-house developed POC device (PATHPOD) that monitored the LAMP reaction by turbidity. The turbidity detection principle of PATHPOD is based on the variation in the light transmittance as a result of precipitation of the magnesium pyrophosphate formed during the amplification of DNA. Next, green light (535 nm) is sent from a LED through the sample, and a phototransistor is used to measure the intensity of transmitted light .Schematic diagram of the electronic circuit board of the fPOC system with a single sample detection that was designed based on Arduino platforms is shown in https://www.parallax.com/package/plx-daq) that allows transferring of collected data from Arduino-Controller board Mega 2,560  to a PC and processing directly in excel format. Then, the received data can be processed, plotted, or graphed, as well as read/write on a worksheet in real-time using Microsoft Excel.Moreover, the PLX-DAQ is a freely downloadable software. The software enabled the computer to receive and process the data generated in the fPOC system. Next, PLX-DAQ is an add-on tool for Microsoft Excel . In early initiated experiments, five different blue LEDs at different center of emitting wavelength, emitting angle, and maximum luminescence are selected and tested . To furt0, 10−1, 10−2, and water  in presence of 5 µM of SYTO-9 dye was used. The results showed that the rRT -LAMP product at 10−2 dilution demonstrated fluorescence intensity of 29,350 arbitrary unit in comparison to 1,850 arbitrary unit observed with the water  were tested in the fPOC system with two different optic filter sets: the Omega and the Optolong Optics filters. In positive reactions, the fluorescence intensity increased quickly after 10 min of the rRT-LAMP reaction, while it remained unchanged in the negative reactions with both filter sets . Howeverin-house POC system (mentioned above). A serial 5-fold dilution of the plasmid control demonstrating a starting concentration of 48.000 copies/reaction was prepared and used as a target for the assay. Linear curves were observed in all three tested systems for the serial dilutions of plasmid control (t 18.34 ± 2.32 min) and qPCR Mx3005P system (Tt 13.50 ± 1.54 min). However, sensitivity of the turbidity-based PATHPOD system was 5 times less sensitive than the two other systems. The Tt value of the PATHPOD system (turbidity detection) was also delayed (Tt 25.46 ± 2.76 min), comparing to the fPOC system (Tt 18.34 ± 2.32 min) and the lab-bench commercial qPCR Mx3005P system (Tt 13.50 ± 1.54 min). The fPOC could detect the SARS-CoV-2 virus at a limit of detection (LOD50%) at 15.36 copies/reaction (two to three copies/microliter) and LOQ at 23.6 copies/reaction (∼4 copies/microliter). The results revealed that the performance of the fPOC system is comparable to the commercial thermocycler qPCR Mx3005P system .The performance of the fPOC system was further investigated using rRT-LAMP assay with the SARS-CoV-2 plasmid positive control as amplification template. The performance of the fPOC was tested and compared to a commercial PCR thermocycler Mx3005P system and PATHPOD, an  control . A LOD oFurther, we further the performance of the fPOC for detecting of SARS-CoV-2 using 12 clinical throat swabs samples that included seven SARS-CoV-2 positive and five negative samples, as confirmed by RT-PCR . The performance of the fPOC was compared to the PATHPOD (turbidity-based) and PCR Mx3005P. Of 12 samples tested, seven samples showed a positive signal, and five samples showed a negative signal in the fPOC platform, PATHPOD system, and qPCR system Mx3005P. The performance of the fPOC platform was in 100% agreement with the commercial PCR MX3005P system, the PATHPOD, as well as with the standard rRT-PCR. Besides purified RNA samples, samples were processed by a simple boiling method and could be directly loaded into the testing cartridges without any additional sample purification steps that minimizes possibility of contamination . As a rehttps://www.pro-lab-direct.com/product-p/gen2-01.htm.). In this study, the new fluorescence-based POC system with estimated material cost of 3.000 $: a ten times lower than the cost of the commercial available is suitable for resource-limited setting for rapid, sensitive, and low-cost pathogen diagnostic.In current times, rRT-LAMP based on both turbidity detection and fluorescence is in practice, since they can monitor the specific gene expression or concentration of target . A commeIn conclusion, we developed and successful proved a concept for a new fluorescence-based POC system for rRT-LAMP. The fPOC is optimized by using different excitation LED light sources, filters, and implemented with Op-amp. The performance of the developed fPOC system for rRT-LAMP was tested, evaluated, and compared with another commercial PCR thermocycler Mx3005P, and PATHPOD system, an in-house developed turbidity detection LAMP based POC system. To detect SARS-CoV-2 in clinical samples, the newly developed fPOC system was used, and the results revealed that the performance of the fPOC system is comparable to the commercial PCR thermocycler Mx3005P system. Moreover, the special design of the fPOC with pyramid optical structures to reflect the light into four different reaction wells, as well as the location of all the integrated components on the top of the reaction well, will reduce the number of the light sources needed and enhance the temperature transfer into the bottom of the reaction and open more opportunity for integrated other functions or components (for example sample preparation). To conclude, the use of open-source hardware/software commercially available Arduino Platforms can provide a promising potential for quick and low-cost design/fabrication of a POC device tuning to attain required analytical sensitivity comparable to commercial systems.


Rectal cancer (RC) is one of the most common malignant tumors. Ferroptosis is an iron-dependent form of cell death, which plays an important role in various cancers. However, the correlation between ferroptosis-related genes (FRGs) and prognosis in RC remains unclear.Gene expression data from The Cancer Genome Atlas Rectum adenocarcinoma (TCGA-READ) and GSE87211 were downloaded. Clustering and functional enrichment were evaluated. A FRGs risk score was established based on the univariate Cox analysis and the Least absolute shrinkage and selection operator (LASSO) analysis. K-M analysis and ROC analysis were conducted to determine prognostic values. qRT-PCR was performed to validate levels of mRNA expression. Multivariate Cox analysis was used to build a prognostic prediction model based on the risk score.P < 0.001), and ROC curve analysis confirmed the signature’s predictive capacity. Multivariate analysis demonstrated that the risk score and age were independent prognostic factors.Based on FRGs, RC patients were grouped into two clusters. In the functional enrichment of differentially expressed genes between the two clusters, immune-related pathways dominated. A novel FRGs signature with 14 genes related to the overall survival (OS) of RC was established. qRT-PCR of the 14 genes identified TP63, ISCU, PLIN4, MAP3K5, OXSR, FANCD2 and ATM were overexpressed in RC tissue; HSPB1, MAPK1, ABCC1, PANX1, MAPK9 and ATG7 were underexpressed; TUBE1 had no difference. The high-risk group had a significantly lower OS than the low-risk group (A novel FRGs model can be used to predict the prognosis in RC, as well as to guide individual treatment.The online version contains supplementary material available at 10.1186/s12864-022-08996-6. Colorectal cancer (CRC) is the third most prevalent cancer worldwide, and rectal cancer (RC) accounts for approximately 40% of all CRCs . RC is tDue to the popularization of digestive endoscopy and the development of MRI, endoscopic ultrasound and other technologies, early diagnosis of RC has been greatly improved , 4, and Some molecular biological diagnostic techniques have been applied to diagnose, treat and predict the prognosis of RC, such as monitoring circulating tumor DNA (ctDNA) expression levels to predict the prognosis , detectiFerroptosis is an iron-dependent programmed cell death pattern characterized by the accumulation of lipid peroxides, proposed by Stockwell et al. in 2012 . The cor− system [The clinical application of ferroptosis focuses on targeting the expression of some FRGs to specifically induce ferroptosis in tumor cells, thus obtaining anti-cancer effects , 18. In − system ; CDKN2A − system . Some st− system , 22. HowIt is necessary for us to analyze FRGs in RC, find ferroptosis-related markers, and evaluate their prognostic value, so as to provide reference for further basic research and clinical translation.https://portal.gdc.cancer.gov/) contains 167 rectum adenocarcinoma (READ) tumor samples and GSE87211 acquired from Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) which include 196 RC patients were used as training set and external validation sets, respectively. We obtained human FRGs from the FerrDB database (http://www.datjar.com:40013/bt2104/). The Human Protein Atlas (HPA) (https://www.proteinatlas.org/about/download) was used to verify the immunohistochemical (IHC) staining of genes.The Cancer Genome Atlas (TCGA) . Volcano plots and a Venn diagram were drawn with the ggplot2 R package. The heat map was generated by the pheatmap R package.The ConsensusClusterPlus R package  was usedGO analysis, including biological process (BP), cellular composition (CC) and molecular function (MF), was performed for FRDEGs characteristics using the clusterProfiler R package . We alsop-value was set at 0.05. Then, least absolute shrinkage and selection operator (LASSO) regression was performed to further screen relevant key genes. Ten-fold cross-validation was used to select the most suitable model parameters λ. Next, we used these key genes to calculate a risk score, which was obtained using the formula: risk score = (βA × Gene A expression) + (βB × Gene B expression) … + (βN × Gene N expression). According to the median risk score, RC patients were divided into two groups: the low-risk group and the high-risk group. The survival differences between the low- and high-risk groups were compared through Kaplan-Meier (K-M) analysis. In addition, we also performed receiver operating characteristic (ROC) analysis to access the survival predictive ability of this risk score. The GEO dataset was used as a validation group to verify the above outcomes.The univariate Cox regression analysis was performed to determine the FRGs with the prognostic value, and the cutoff −ΔΔCt) method, and the mean value was used as the final experimental result for replicated wells. All procedures were carried out in accordance with the manufacturer’s instructions.qRT-PCR was performed on 10 pairs of RC tissues and adjacent normal tissues to validate the mRNA expression levels of the 14 signature genes. Consent forms were obtained from each patient for the collection and analysis of tissue samples. The study was approved by the Peking Union Medical College Hospital ethics review boards. We immediately froze and stored the tissues in liquid nitrogen after extracting them. Total RNA was extracted from the sample tissues via Trizol lysate (Thermo Fisher Scientific), followed by reverse transcription to cDNA. qRT-PCR was carried out using the CFX96 system . GAPDH served as an internal control. Relative expression levels were quantified by the Ct . Pathways with an adjusted Evaluation of immune microenvironment characteristics, immune checkpoint related characteristics and chemotherapy drugs’ sensitivity.http://tide.dfci.harvard.edu/) algorithm was used to estimate the ICIs response of RC patients from TCGA cohort [xCELL  algorithA cohort . The pRRA cohort  was usedUnivariate and multivariate Cox regression analyses were performed to establish the prognostic model based on the risk score and other clinicalpathological characteristics (age and TNM stage) of the patients. We constructed a nomogram to predict the overall survival (OS) of RC patients in 1-, 3- and 5-year according to the prognostic model. Then, we used the median total points for each patient based on this nomogram, to divide patients into high-risk and low-risk groups. The K-M curve was plotted and ROC analysis was carried out at 1-, 3- and 5-year of survival. Next, calibration curves were used to estimate whether the predicted survival results of the nomogram  were close to the actual results.https://www.r-project.org/) and its appropriate packages. Univariate, LASSO and multivariate Cox regression analyses were conducted to build models. Log-rank test was used in the K-M analysis. Two groups were adjusted by the Wilcoxon test and the p-value was corrected by Benjamini and Hochberg (BH) test. Two-sided P < 0.05 indicated statistical significance.All analyses were performed using R version 4.1.3 . Visualization of the two groups after dimensionality reduction was shown in Fig. p-value) in BP, CC and MF respectively. Differential genes were enriched in immune-related pathways in each facet, and were additionally associated with phagocytosis, transmembrane transport, GABA receptors, ion channels etc. The result of the KEGG enrichment analysis was shown in Fig. The results of the GO enrichment analysis were presented in Fig. P < 0.05)  with a hazard ratio (HR) of 8.36 (95%CI 3.95–17.7)  Fig. . To prev05) Fig. . When th05) Fig. . The ris05) Fig. . The K-M.7) Fig. . The are.7) Fig. .Fig. 4ThP = 0.0381) with a HR of 2.26 (95%CI 1.08–4.74) . The distribution of risk scores and survival status in the dataset is presented in Fig. 74) Fig. . The AUC74) Fig. .Fig. 6a To further confirm the importance of the 14 FRGs in the risk score, HPA database was used to compare their protein expression in non-cancerous and RC tissues. According to Fig. In RC tissue, the expression of TP63, ISCU, PLIN4, MAP3K5, OXSR, FANCD2 and ATM was significantly higher than that of normal tissue. In contrast, the expression of HSPB1, MAPK1, ABCC1, PANX1, MAPK9 and ATG7 was significantly lower than that of normal tissue. There was no significant difference in the expression of TUBE1. Fig. .Fig. 8ThThe pathways of ribosomes, oxidative phosphorylation, and arachidonic acid metabolism were mainly enriched in high-risk groups. In contrast, the pathways of DNA replication, homologous recombination, RNA degradation, cell cycle and spliceosome were mainly enriched in the low-risk group Fig. .Fig. 9GS+ Tem, CD8+ naive T-cells, mast cells, monocytes, NK cells, Tgd cells, Th2 cells were significantly different  Fig.  in the tRC is a dangerous malignant tumor that threatens human health. At present, the efficacy of comprehensive treatment needs to be improved. Many molecular mechanisms are still being explored, and many reasons for the occurrence and development of RC are still unclear. Ferroptosis is an iron-dependent form of cell death. In the field of malignant tumors, people hope to inhibit tumor development by promoting ferroptosis in tumor tissue, and providing new targets and a pharmacological basis for tumor therapy.In this study, the risk score of FRGs in RC was established by analyzing the transcriptome, clinical information, and survival information of TCGA READ. A risk prediction model was built on the basis of the risk score, combined with multivariate Cox analysis. The model can effectively predict the 1-, 3- and 5-year OS of RC, providing a potentially useful tool for clinical practice and further mechanism research.First, we performed a cluster analysis on TCGA READ samples from the perspective of FRGs. The clustering results were divided into two categories, meaning that the RCs were divided into two different types of ferroptosis gene expression patterns. We use GO enrichment analysis and find that these FRDEGs are mainly enriched in immunity in BP, CC and MF. This suggests that the differences in gene expression of ferroptosis in RC patients are closely related to immune responses. Many studies have shown that ferroptosis is closely associated with the tumor immune microenvironment . The stuTo further investigate FRGs affecting RC, univariate Cox regression was applied to screen genes significantly associated with OS. Then, 14 genes were identified using LASSO regression to establish a risk score. We divided patients into the high- and low-risk groups by the risk score, and survival analysis indicated that the prognosis of the high-risk group was significantly worse. ROC analysis showed that this risk score was very accurate in predicting OS at 1, 3, and 5 years in the TCGA READ cohort. Therefore, we selected another RC cohort from GEO for external validation. The results showed that there was still a significant difference in OS between the two groups. The accuracy of OS prediction, though decreased, was still above 0.6, indicating that the risk score has reasonable extrapolation ability. We briefly checked the expression of the 14 genes encoded proteins through the HPA database, and found that only ATM was significantly highly expressed in RC tissue. Then, we performed qRT-PCR analysis of these 14 genes with surgical specimens from our research center, and the results showed that TP63, ISCU, PLIN4, MAP3K5, OXSR, FANCD2 and ATM were significantly highly expressed in tumor tissue. The reasons for the different results of IHC and qRT-PCR include: the sensitivity of IHC is not high, resulting in the failure to identify proteins with significant differences; there are some additional intervening factors in the process from transcription to translation, which need to be further explored.We briefly introduce these 14 genes one by one. TP63 encodes a member of the p53 transcription factor family, and studies have shown that up-regulation of TP63 may further activate the glutathione metabolic pathways, including GPX4, thus reducing cellular exposure to oxidative stress, which supports the survival of tumor cells , 34. ISCWe further analyzed the underlying mechanisms of different prognosis between high- and low-risk groups. The GSEA results indicate that the enrichment pathways of the two groups are different. The high-risk group tends to be enriched in energy metabolism such as ribosomes, oxidative phosphorylation, and arachidonic acid metabolism. High-risk patients are likely to require the joint participation of the above pathways due to the inhibition of ferroptosis. Analysis of both groups of immune microenvironments found that, from the perspective of the ESTIMATE algorithm, the risk score was not correlated with the total number of immune cells and stromal cells. However, there are significant differences in the number of different types of immune cells in high- and low-risk groups from the perspective of the xCELL algorithm, and the high-risk group has significantly more CD8+ naive T cells and Th2 cells. In CRC, a substantial density of CD8+ T-cells in tumor tissue is generally associated with a favorable prognosis and sensitivity to chemoradiotherapy and immunotherapy , 50, par4EBP1/eIF4E/PUMA pathway [Combined with the analysis of drug susceptibility in the database, it was suggested that patients in the high-risk group were more sensitive to certain drugs. Although the difference in IC50 was not too large, it still provided us with an idea that these drugs may inhibit RC by enhancing ferroptosis. AS605240 is a kind of phosphatidylinositol 3-kinase (PI3K) gamma inhibitor. Studies have shown that oral administration of AS605240 in a mouse colitis model can reduce intestinal inflammation in mice through a number of immunomodulatory effects . MK-2206 pathway . At pres pathway . It has  pathway . Therefo pathway , so this pathway . It was  pathway . A recen pathway . Therefo pathway .To further improve the clinical predictive value of the risk score, we combined common clinical prognostic factors (age and TNM stage) to construct a final prognostic model, which is composed of the risk score and age. 1-year, 3-year and 5-year OS were predicted, and the prediction accuracy was further improved compared with the risk score alone. We look forward to further discussion and promotion of the detailed mechanism of this model. In recent years, many clinical prognostic models have been built, involving various cancers and mechanisms , 64–66. This study provides a risk score of FRGs for RC by analyzing the TCGA READ cohort from the perspective of FRDs, which can effectively distinguish the prognosis of two groups of patients and predict 1-year, 3-year and 5-year OS. The 14 genes involved in this risk score may affect the occurrence and development of RC. It may also affect drug sensitivity from ferroptosis, immune infiltration and other aspects, providing a reference for further in-depth discussion. We constructed a more complete prognostic model combined with the clinical characteristics of the patients, which enhanced the predictive ability of the risk score.Additional file 1:Fig. S1. (A) Consensus matrix legend of the cluster. (B-I) The heatmap of consensus matrix with k from 2 to 9. (J) Consensus cumulative distribution function (CDF). (K) Relative change in area under CDF curve. (L) Tracking plot of the cluster.